The need for replacement tissues and/or organs for the human body, in combination with the shortage of donors has been a strong incentive for the development and production of tissue engineered implants that can take over the function of missing or injured body parts. An advantage of these “engineered” replacement tissues and organs is that they may circumvent many of the hazards and problems associated with donor tissues and organs, and at lower cost. Today, tissue engineering (TE) applications cover virtually every human tissue, including skin, eyes, liver, pancreas, blood vessels, ligaments, cartilage, bone, muscle and parts of the nervous system.
The principle of TE is relatively simple and involves the isolation and culture of cells, and the seeding of the cultured cells onto a biological or artificial scaffold in vitro prior to transplantation of the seeded scaffold into the specific location of the body. The scaffold thereby serves as an attachment matrix and guides the cells during tissue formation or regeneration. The regeneration of the seeded cells into a tissue may take place prior, during or after the implantation. The cells that are used for regenerating a tissue should exhibit an inherent regenerative capacity. Stem cells, that have the ability to differentiate into a variety of different cell types, are now commonly used for this purpose. Stem cells are undifferentiated progenitor cells and may for instance be isolated from autologous sources (patient's bone marrow) and expanded in in vitro culture. Preferred progenitor cells are bone marrow derived mesenchymal stem cells (MSC), that may form connective tissues such as bone, cartilage, tendon, ligament, bone marrow stroma, mucous tissue, fat and muscle. These progenitor cells can be induced by specific bioactive molecules to mature into a required cell type. MSCs may for instance be induced to form osteoblasts by using dexamethasone. Once matured, the cells are then seeded onto the scaffold.
Prior to the implantation of the seeded scaffold in vivo, the seeded cells can be induced by yet other specific bioactive molecules such as growth factors, by ex-vivo gene transfer or by other physical factors to form the required neotissue in vitro. Alternatively, the scaffold comprising the growth factors may be combined with the cells in vivo. An example of the first TE procedure is that of tissue engineering of bone, wherein bone-marrow derived mesenchymal stem cells are expanded, differentiated to bone-forming osteoblasts and subsequently seeded on a biodegradable scaffold. The scaffold is osteoconductive in that it provides a path for the growing bone tissue. The in vitro prepared and loaded scaffold is then implanted in a bone defect and while the seeded cells are induced to form new bone material, the scaffold itself is degraded. An example of the latter TE procedure is that of human articular cartilage repair using the patient's own autologous chondrocytes retrieved at arthroscopy. The chondrocytes are expanded in vitro before being reimplanted into full-thickness articular cartilage defects covered with a sutured and fibrin-glued periosteal patch. Of course, tissues or organs may also be produced completely in vitro and transplanted as ready replacement materials.
Though simple in conception, these procedures can be quite complex in practice. While TE of skin is relatively straightforward due to the 2-D arrangement of cells, the formation of more complex solid structures such as bone and even complete organs is far more demanding, even under controlled in vitro conditions. Bone formation itself preferably occurs in situ since proper bone formation requires that bone is formed in the direction of the functional pressures and is highest in density at high-pressure sites. The compact and spongy material of natural bone is composed such that maximum strength is produced with a minimum of material and, in form and structure, must be formed such that the maximum compressive stresses normally produced by the body weight are resisted in the most economical manner. Moreover, natural formation of bone is the result of the combined action of bone-forming osteoblasts and bone degrading osteoclasts, and the flow of interstitial fluids is believed to play an important role in the activation of the various processes (Hillsley and Frangos, 1994). As a result, functional bone formation cannot properly occur in vitro.
An important problem with in vitro produced TE implants (grafts) is that once the grafts are transplanted in vivo the nutrient supply to the cells is discontinued. Tissues of any size need a blood supply to bring in nutrients and carry out dissimilation products. During the first 6-8 days post transplantation, the vascularization of the implant is the principal factor that limits the viability of the cells. This problem can only partially be addressed by using matrices that house growth factors to stimulate blood vessel ingrowth from the surrounding tissue. Substituting these growth factors with blood-vessel progenitor cells that sprout vessels from within the body of the matrix is one way of enhancing the rate of vascularization. Internally derived vessels then only need to link with surrounding vessels to establish a flow-through circulation. However, because such processes are slow, many of the cells in the implant will have died by then and tissue ingrowth (i.e. ingrowth into the scaffold as to form the 3-D tissue) will be severely hampered.
The possibility of limited tissue ingrowth in tissue-engineered constructs due to insufficient nutrient transport is an important concern in tissue regeneration. Research into the flow velocity around and through scaffolds, as performed with specialized high-aspect-ratio vessel rotating bioreactors and complex three-dimensional (3D) scaffolds for culturing osteoblast cells (Yu et al., 2004), has revealed that the 3D dynamic flow environment affects bone cell distribution, cell phenotypic expression and mineralized matrix synthesis within tissue-engineered constructs. Such studies are important for the design and optimization of 3D scaffolds suitable in bioreactors for in vitro tissue engineering of bone and stress the need for a proper flow of fluids through the engineered tissue.
Another important problem associated with tissue regeneration in TE is associated with biodegradable scaffolds. Although not exclusively, TE techniques generally involve the use of a temporary biodegradable scaffold that serves as 3D template for initial cell attachment and subsequent tissue formation. The ability of the scaffold to be metabolized by the body allows it to be gradually replaced by new cells to form functional tissues. However, these biodegradable scaffolds also have their downside. Without proper removal of degradation products, accumulation of waste products may impede the growth of the tissue-regenerating cells. This problem can only partially be solved by the use of scaffolds with a slow degradation rate and often requires the use of non-degradable permanent scaffolds.
Thus, in general, cell growth on biodegradable scaffolds requires high levels of mass transfer. Proper growth requires the transfer of sufficient amounts of nutrients and oxygen to the tissues and degraded scaffold mass must be removed. Production of tissue replacements outside the body (ex vivo) is already difficult since conventional bioreactor devices with impeller mixers are often not effective for providing mixing and mass transfer. This challenge arises due to the lack of rapid vascularization (angiogenesis) of large three-dimensional (3-D) scaffold constructs.